The use of bacterial extracts for generating, proteins of interest m cell free protein synthesis reactions (CFPS) has been expanding from a laboratory scale to commercial scale. There is a continuing need to make the process more economical. A part of this need includes improved methods for storing and stabilizing bacterial extracts. Prior art methods included the use of various additives; freezing and thawing; spray-drying or conventional lyophilization. Drying bacterial extract has obvious advantages of reduced volume but typically resulted in significant reduction in the ability of the extracts to generate the proteins of interest.
In conventional freeze-drying (lyophilization) of bacterial extracts, vacuum pressure lowers the boiling temperature of water in the extract and enables the bacterial extracts to be dried at a lower temperature. The process reduces thermal degradation, relative to higher temperature drying procedures. Typically a pressure gradient is created within the extract, driving mass transfer as vaporization occurs, and increasing heat transfer such that a heat gradient forms. Due to the heat gradient, outer portions of the sample tend to dry first, causing the surface layer to become more isolating for interior regions of the material and preventing more rapid heat transfer. Conventional lyophilization can lower the viability of biologically-active materials in bacterial extracts.
In situ vaporization provides an expansive force in the sample as water vapor escapes, maintaining a more porous structure with increased surface area for sublimation. Microwave radiation in a vacuum chamber (radiant energy vacuum) is used to remove water to dehydrate biological materials (e.g., proteins, enzymes, nucleic acids, macromolecules, etc.). In some cases, biological material is frozen at low temperature in a vacuum chamber and radiant energy applied, such that the ice in the material is sublimated. In other cases the biological material is frozen prior to entering the vacuum chamber. This microwave radiation sublimation process prevents damage to temperature-sensitive biological materials from elevated temperatures and increases the drying rate and decreases drying times, relative to conventional freeze-drying procedures.
Formulations of liquid extract prior to freeze drying enable integration of non-volatile additives into the dried extract. The presence of additive(s) has been shown to improve or increase the extract stability during storage. For example, an inositol can be added to wheat germ extracts for use in cell free protein synthesis (see, U.S. Pat. No. 7,048,915).